The primary degradative compartment in the cell is the lysosome (or its equivalent in plants and yeasts, the vacuole). Although cells have evolved multiple mechanisms to deliver cellular components to the lysosome, the major route is via a set of processes collectively termed autophagy. Frequent targets of these processes are peroxisomes, organelles that contain essential oxidative enzymes. The number, size, and enzymatic content of peroxisomes change rapidly in response to a cell's metabolic needs, and when peroxisomes of a certain metabolic type are no longer useful, they are selectively removed by autophagic processes. The goal of this project is to understand the molecular mechanisms that control autophagy. A genetic approach has been initiated using the yeast Pichia pastoris as a model system. This yeast has large numerous peroxisomes when cultured on methanol, which are selectively autophagized upon transfer to either glucose or ethanol. During the period of our previous FIRCA, we succeeded in isolating and characterizing P. pastoris mutants in six different genes that are defective in autophagic peroxisome degradation in response to glucose. Four of these PDG genes were isolated, and DNA sequencing results indicate that all four encode novel proteins of unknown function. The specific aims of this continuing FIRCA proposal are: (1) to characterize the four PDG proteins by determining their subcellular location and searching for other genes whose products interact with them; (2) to determine what other vacuolar sorting pathways are affected by mutations in each gene; and (3) to develop and utilize novel screens and selection methods for the isolation of mutants in new PDG genes. Results of these studies will provide important insights into autophagic mechanisms.